Tumor-Specific Chromosome Mis-Segregation ControlsCancer Plasticity by Maintaining Tumor HeterogeneityYuanjie Hu1, Ning Ru2, Huasheng Xiao3, Abhishek Chaturbedi2, Neil T. Hoa4, Xiao-Jun Tian5,
Hang Zhang5, Chao Ke2,6, Fengrong Yan2, Jodi Nelson2, Zhenzhi Li2, Robert Gramer2, Liping Yu7,
Eric Siegel8, Xiaona Zhang3, Zhenyu Jia9,10,11,12, Martin R. Jadus4,12, Charles L. Limoli13, Mark E. Linskey2,
Jianhua Xing5*, Yi-Hong Zhou1,2*
1 Department of Biological Chemistry, University of California Irvine, Irvine, California, United States of America, 2 Department of Neurological Surgery, University of
California Irvine, Irvine, California, United States of America, 3 National Engineering Center for Biochip at Shanghai, Shanghai, China, 4 Diagnostic & Molecular Health Care
Group, Veterans Affairs Medical Center, Long Beach, California, United States of America, 5 Department of Biological Sciences, Virginia Polytechnic Institute and State
University, Blacksburg, California, United States of America, 6 State Key Laboratory of Oncology in South China and Collaborative Innovation Center for Cancer Medicine,
Sun Yat-sen University Cancer Center, Guangzhou, China, 7 Ziren Research LLC, Irvine, California, United States of America, 8 Department of Biostatistics, University of
Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America, 9 Guizhou Provincial Key Laboratory of Computational Nano-Material Science, Guizhou
Normal College, Guiyang, China, 10 Department of Statistics, University of Akron, Akron, Ohio, United States of America, 11 Department of Family and Community
Medicine, Northeast Ohio Medical University, Rootstown, Ohio, United States of America, 12 Department of Pathology & Laboratory Medicine, University of California
Irvine, Irvine, California, United States of America, 13 Department of Radiation Oncology, University of California Irvine, Irvine, California, United States of America
Abstract
Aneuploidy with chromosome instability is a cancer hallmark. We studied chromosome 7 (Chr7) copy number variation(CNV) in gliomas and in primary cultures derived from them. We found tumor heterogeneity with cells having Chr7-CNVcommonly occurs in gliomas, with a higher percentage of cells in high-grade gliomas carrying more than 2 copies of Chr7,as compared to low-grade gliomas. Interestingly, all Chr7-aneuploid cell types in the parental culture of established gliomacell lines reappeared in single-cell-derived subcultures. We then characterized the biology of three syngeneic gliomacultures dominated by different Chr7-aneuploid cell types. We found phenotypic divergence for cells following Chr7 mis-segregation, which benefited overall tumor growth in vitro and in vivo. Mathematical modeling suggested the involvementof chromosome instability and interactions among cell subpopulations in restoring the optimal equilibrium of tumor celltypes. Both our experimental data and mathematical modeling demonstrated that the complexity of tumor heterogeneitycould be enhanced by the existence of chromosomes with structural abnormality, in addition to their mis-segregations.Overall, our findings show, for the first time, the involvement of chromosome instability in maintaining tumorheterogeneity, which underlies the enhanced growth, persistence and treatment resistance of cancers.
Citation: Hu Y, Ru N, Xiao H, Chaturbedi A, Hoa NT, et al. (2013) Tumor-Specific Chromosome Mis-Segregation Controls Cancer Plasticity by Maintaining TumorHeterogeneity. PLoS ONE 8(11): e80898. doi:10.1371/journal.pone.0080898
Editor: Anita B. Hjelmeland, Cleveland Clinic, United States of America
Received May 30, 2013; Accepted October 17, 2013; Published November 25, 2013
Copyright: � 2013 Hu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by UC Irvine set-up funds and a generous Stern Family gift (YHZ and ML), grants provided by UC Cancer ResearchCoordinating Committee, UC Irvine Committee on Research, Cancer Center Seed Grant (Award Number P30CA062203 from the National Cancer Institute), MusellaFoundation for Brain Tumor Research & Information, and Voice Against Brain Cancer (YHZ), National Science Foundation DMS-0969417 (JX), VA Merit ReviewGrant (MRJ). Zhenyu Jia is partially supported by Guizhou Normal College, Guiyang, Guizhou, China, QKHJ[2013]2238. The funders had no role in study design,data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Liping Yu is an employee of Ziren Research LLC, Irvine, CA, USA. This does not alter the authors’ adherence to all the PLOS ONE policieson sharing data and materials.
* E-mail: [email protected] (Y-HZ); [email protected] (JX)
Introduction
According to Nowell’s initial clonal evolution hypothesis [1],
cancer development is an evolutionary and ecological process, in
many ways resembling Darwinian evolution [2]. This hypothesis is
supported by prediction of tumor progression with genetic clonal
diversity in esophageal adenocarcinoma [3], and now has been
widely accepted as an explanation for the tumor heterogeneity
observed in most cancers at the time of clinical diagnosis, at both
the original and metastatic sites [4,5]. The concept of cancer as an
evolutionary process, with tumors having genetically and pheno-
typically diverse cell subpopulations is consistent with the recent
cancer stem cell model, which emphasizes the importance of
cancer having a cell type capable of generating other cell types in a
unidirectional manner [6–9]. However, the finding of phenotypic
inter-conversion among three subpopulations of cells within breast
cancer cell lines, leading to a cell population equilibrium [10]
revealed the ability of cancer to recover biological diversity from
more than just the stem-like cell subpopulation. Such ability to
recover equilibrium conditions after a disturbance is a feature
characteristic of an established, well-balanced ecosystem. The
question remains whether, and how, cancer cell phenotypic
transition manifests as an inherited feature.
Accumulating evidence supports the notion that mitotic errors
cause chromosome instability, which drives cancer evolution, with
natural selection acting at the cancer ecology level to avoid
cytogenetic chaos. Apparently, the non-random distribution of
chromosomal gains and losses seen in specific tumor types is a
PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e80898
combined effect of chromosome instability and selection for
specific phenotypes from among massive changes of the tran-
scriptome [11–15]. Gliomas are primary malignant brain tumors
having astrocytic and/or oligodendroglial features of varying
malignancy. The highest grade, unfortunately the most commonly
seen glioma, is glioblastoma multiforme (GBM, grade IV),
morphologically, genetically, and cytogenetically heterogeneous,
and uniformly fatal due its rapid cellular proliferation and strongly
invasive behavior [16–19]. It is known that alteration of
chromosome 7 (Chr7) copy number occurs in both high- and
low-grade gliomas and that these changes appear to be associated
with invasive and proliferative cell phenotypes [20–24]. Here we
report studies of Chr7-aneuploidy-related cell diversity and the
role of Chr7 mis-segregation (Chr7-MS) in maintaining the
phenotypic diversity of glioma cell subpopulations, which gener-
ates a synergistic effect on overall tumor growth.
Materials and Methods
Ethics StatementFrozen and fresh glioma specimens were provided by the Tissue
Banks of University of California, Irvine and University of
Arkansas for Medical Sciences, with Institutional Review Board
approval.
Animal work and subcutaneous (s.c.) and intracranial(s.c.) xenografts
The animal work was approved by Animal Care and Use
Committee (IACUC) of University of California, Irvine. For
studies using intracranial (i.c.) xenografts, glioma cells (16105/3 ml
DMEM/F12) were injected into the frontal lobe of 4–6 week old,
female, nude mice (stain NCrNu-M, Taconic, Hudson, NY),
following IACUC approved surgical procedures. After i.c.
implantation, mice were observed daily and periodically weighed
for moribund signs (hunchback posture, marked weight loss and
gait impairment). Mice were euthanized when they developed
brain-damage symptoms (ataxia, hemiparesia, etc) and/or 20%
body weight loss, and the following day was record as the survival
date for survival analysis.
For studies using subcutaneous (s.c.) xenografts, cells (16106
cells/50 ml DMEM/F12) were subcutaneously injected into nude
mice, anterior to their right and left thighs, on both sides. Tumor
measurements were taken every 3–4 days after implantation, and
tumor volume was calculated using the formula V = (L*W2)/2 (L,
length; W, width). Mice were euthanized at a predetermined time
of the experiment or when tumor volume exceeded 1.5 cm3.
Glioma primary cultures and cell linesFresh human glioma tissues were dissociated enzymatically
(0.05% trypsin-EDTA for 30–45 min at 37uC), disrupted
mechanically (passing through a glass pipette in DMEM/F12
containing 0.10 mg/ml DNase and 10% serum), and cultured in
both collagen-coated (3–4 mg/cm2) culture dishes in DMEM/F12
supplemented with 5% fetal bovine serum, designated as serum
adherent (SA) culture conditions, and agar (1%)-coated culture
dishes in DMEM/F12 supplemented with epidermal growth
factor (EGF, 20 ng/ml), basic fibroblast growth factor (FGF,
10 ng/ml), and 1–5% B27 (Invitrogen, Carlsbad, CA), designated
as neural sphere (NS) culture conditions. The multicellular glioma
spheres formed in NS culture conditions were passed into
fibronectin (1 mg/cm2) coated dishes in the same culture medium
before freezing or subjecting to FISH analysis.
The human glioma cell lines (A172, LN229, LG11, T98G,
U251, and U87) were obtained from the Department of Neuro-
Oncology, the University of Texas M.D. Anderson Cancer
Center. The genetic profiles (7-STR markers provided by IDEXX
RADIL, Columbia, MO) used by this study were identical or
highly similar to the genetic profiles reported for each cell line
(Table S1). A172 reported here was originally named as D54, but
carries genetic profiles suggesting a variant of A-172 reported by
American Type Culture Collection (ATCC). U251 reported here
was originally named as U251HF, with genetic profile suggesting a
variant of U251, compared to U251 in NCI-60 Cancer Cell Line
Panel. The comparisons of U251 variants (Table S2) were
provided by Beth Bauer (IDEXX RADIL).
All glioma cell lines (parental) were cultured in SA conditions.
The derived SA and NS clones were established from single
colonies formed in 0.3% soft agar on top of a layer of bottom agar
(0.5%) in DMDM/F12 supplemented with 5% bovine serum or
EGF/bFGF/B27 as for NS cultures, picked by a glass pipette, and
expanded in SA or NS conditions. For U251 the same
homozygous mutations of PTEN [E242fs*15 (723 724 insTT)]
and TP53 (R273H) in parental and SA and NS-subcultures were
identified by Mariam Youssef, Nirvi Shah and Anthony Wong
(UC Irvine).
Fluorescence in situ hybridization (FISH)Metaphase-spread slides were obtained by exposing 80%
confluently growing cells to nacadozole solution (100 mg/ml final,
Sigma) for 1 hour. Then the cells were trypsinized (0.25% trypsin/
EDTA, Invitrogen) to collect cell pellets, which were treated with a
hypotonic solution (phosphate buffer) for 5 minutes at 37uC. The
cell pellets were fixed (methanol:glacial acetic acid = 3:1) for at
least 30 minutes. Finally, the cell suspensions were dropped onto
slides to get metaphase chromosome spreads. The standard B-
banding was done for the slides as they were done as (same time)
treatment with trypsin, and stained with Giemsa stain (Invitrogen).
FISH was performed on metaphase spreads and frozen tumor
sections (7 mm) using Direct Labeled Fluorescent DNA Probe Kits
with CEP X/CEPY, EGFR/CEP 7 and PTEN/CEP10 (Abbott
Molecular Inc. Des Plaines, IL). Hybridization, washing, and
counterstaining were performed according to the manufacturer’s
instructions. 250–300 cells per sample were counted under a
fluorescent microscope with a 1006lens.
Lentivirus infectionInfectious lentivirus was produced by co-transfection of the
lentiviral vector plasmid pGIPZ-Empty and pTRIPZ-Empty
(Open Biosystems) with packaging plasmid psPAX2 and envelope
plasmid pCMV-VSVG in HEK-293T cells, following the manu-
facturer’s protocol.
Immunofluorescence analyses of i.c. xenograftsThe cryosections (7–8 mm) of mouse brains with i.c. xenografts
were mounted for direct observation of fluorescence expressed by
the RFP and GFP-labeled tumor cells using 26and 206 lenses of
a Keyence BZ8100 fluorescence microscope, after nuclear staining
with DAPI. Adjacent cryosections were subjected to immunoflu-
orescence analyses using 15 mg/ml rabbit BMI1 (ab38432,
Abcam), 15 mg/ml mouse CD133 (130-090-422, Miltenyi Biotec),
10 mg/ml mouse CD31 (CBL1337, Chemicon), 15 mg/ml goat
GFAP (sc-6170, Sana Cruz), 10 mg/ml rabbit MELK (A01390,
GenScript), and 15 mg/ml mouse SPARC (sc-73051, Sana Cruz)
primary antibodies, followed by appropriate secondary antibody,
donkey anti-mouse, rabbit, rat, or goat Alexa Fluor 350 (blue),
Alexa Fluor 488 (green), and Texas Red (Invitrogen), following the
immunofluorescence process as described previously [25]. The
tissue sections were mounted with ProLong Gold antifade reagent
Chromosome Mis-Segregation and Tumor Heterogeneity
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(Invitrogen), viewed with a 406lens of a fluorescence microscope
and imaged with a spot camera. Co-localization images were
acquired and analyzed using a Nikon two-laser (HeNe and Argon)
PCM 2000 Confocal System on an Eclipse E800 Microscope with
1006 objective (Melville, NY)_ENREF_20.
Neural stem cell differentiation assayGlioma cells (5–106103) maintained in neural sphere culture
conditions were seeded into 8-well slides pre-coated with
fibronectin (10 ng/ml) for overnight culture in original medium
(undifferentiation), or in poly-L-lysine (15 mg/ml) coated wells in
DMEM/F12 containing 1% FBS for 7–10 days of culture
(differentiation); a half volume of fresh medium was added every
3 days, prior to fixation for immunofluorescence analyses. Cells
were fixed with 4% PFA and blocked with 10% donkey serum.
Primary antibodies (rabbit Nestin (1:1000) from Millipore
(AB5922), mouse MAP2 (1:200) from Abcam (ab11267), and goat
GFAP (1:300) from Sana Cruz (sc-6170), mouse Beta Tubulin III
(1:200) from Chemcon (MAB1637)), mouse CD133 (1:50) from
Miltenyi Biotec (130-090-422), rabbit MELK (1:200) from Gen-
Script USA (A01390), and rabbit BMI (1:200) from Abcam were
incubated with cells overnight at 4uC and developed using
AlexaFluor secondary antibodies (mouse or rabbit Alexa Fluor
488 nm and 594 nm (1:200) from Invitrogen).
Real-time comparative quantitative polymerase chainreaction (CQ-PCR) and quantitative reverse transcription(qRT-) PCR
DNA samples from frozen glioma specimens were isolated using
a DNeasy kit (QIAGEN, Valencia, CA). CQ-PCR standard
(product CQ101) and PCR primers to quantify EGFR and three
reference genes in 2q34 (SPAG16), 3p14.3 (ERC2), and 5q31.2
(SPOCK1) were from Ziren Research LLC (Irvine, CA). It is a
recombinant DNA containing PCR fragments of EGFR and
reference genes in one piece to determine CNV as described
previously [26]. Real-time PCR was carried out using FAST-
START SYBR-Green I Master Mix (Roche).
Total RNA (,1 mg) extracted using Ultraspec (Biotecx) from
SA and NS-adherent cultures, after a 24-hour culture in basal
medium, was converted into cDNA using 5 units of Superscript II
reverse transcriptase (Invitrogen). The cDNA samples were diluted
and quantified for gene expressions by real-time qRT-PCR (SYBR
Green I) using a single standard for marker and reference genes
[27], normalized to ACTB. Quantification of GAPDH was also
performed to compare with gene of interest. The primer sequences
for genes in qRT-PCR and CQ-PCR are available from Ziren
Research LLC (www.zirenresearch.com) upon request.
Comparative genome hybridization (CGH)DNA (1.5 mg) samples of glioma cells and control (a pool of six
normal human blood DNA samples) were differentially labeled
with Cy5 and Cy3-dUTP, respectively, purified and then
hybridized to an Agilent Human Genome CGH 244 k Micro-
array. The data were statistically analyzed and visualized using
two independent methods, including Agilent Genomic Workbench
6.5 (Agilent) with Z-score algorithm and a program written in R
(http://www.r-project.org/), which detected the same chromo-
somal aberrations. The threshold of the Z-score used for the
Agilent method was set to 4.
Gelatin zymography, enzyme immunometric assays,Western blotting, and immunocytofluorescence
Proteins in 24-hour conditioned cell culture media were
precipitated with 4 volumes of cold acetone, spun immediately
at 14,000 rpm for 5 minutes at 4uC, and resuspended in
radioimmunoprecipitation assay buffer (RIPA) containing Prote-
ase Inhibitor Cocktail (Roche). The same amount of conditioned
medium protein was used to run gelatin zymography. Conditioned
medium was subjected to enzyme-linked immunosorbent assay
(ELISA) for VEGFA (VEGF-165) and SPP1 (Osteopontin) using
kits from Assay Designs (Ann Arbor, MI), and PTN from R&D
Systems (Minneapolis, MN). Sonicated whole-cell lysate in RIPA
was used to perform Western blotting, with antibodies of EGFR
from Cell Signaling, and Actin from EMD Bioscience. Cells
seeded on Poly-L-lysine or Fibronectin coated 8-well chamber
slides, 26104 cells per chamber, and incubated overnight, were
fixed with 4% paraformaldehyde in PBS, with a brief permeabi-
lization in 0.1% triton x-100, and an overnight incubation with
primary EGFR antibody at 4uC. The immunocytofluorescence
signal was detected after incubation with Alexa FluorH 594
secondary antibody.
Soft agar colony formation assay800–1000 cells were mixed with 1 ml of 0.3% soft agar in
DMEM/F12 supplemented with 5% bovine serum or a mitogen
supplement for NS cultures as detailed above, spread onto
hardened 0.5% soft agar in the same medium (1 ml per well in
four corner wells of a 6-well plate). 1 ml of the same medium was
added 2 and 3 weeks later and colony numbers were counted 4
weeks later under a microscope with 46lens.
Statistical analysisMANOVA analysis was used in conjunction with ternary plots
(http://www.davidgraham.org.uk) to compare GBM to OG
samples for percentages of cells bearing one copy, two copies, or
$3 copies of Chr7. Stem-like cell- and nonstem-like cell-enriched
subcultures were compared for differences in gene expression,
ELISA, and zymography data by means of 2-sample equal-
variance t-tests. Overall survival of mice bearing intracranial
glioma xenografts was estimated via Kaplan-Meier survival curves,
then compared for differences using a stratified Cox regression
model in order to adjust for potential variation (‘‘Day effects’’)
between different experiments. SAS versions 9.2 and 9.3 (The SAS
Institute, Cary, NC) were used for all analyses and P,0.01 was
used as the significance value to adjust for multiple comparisons
without overinflating Type II error.
Mathematical modelingMathematical model Construction. Denote x1, x2, x3, x4,
x5 as the abundances of cells with 1–5 copies of Chr7. We
neglected cells with 6 copies, assuming that they were anaphase
stages of 3-copy Chr7 cells. We didn’t expect that the results below
would be significantly affected by this assumption since the
percentage of the 6-copy cells is low in all the measurements. For
simplicity we also assume that the mis-segregation rates of normal
and abnormal Chr7 are the same. For STICs (2Chr7:1n,1d), we
assume the cells can either divide symmetrically, or have one Chr7
mis-segregation, as summarized below
x2?2x2, no missegregation with rate r2(1{2p2)x2
x1zx3, one missegregation with rate 2r2p2x2
�
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Similarly for TMCs (3Chr7:2n,1d), we have
x3?
2x3,no missegregation or two missegregations with
rate r3(1{3p3{3
2p2
3)x3
x2zx4, one missegregation with rate 3r3p3x3
x1zx5,two missegregations with rate3
2r3p2
3x3
8>>>>>><>>>>>>:
The parameters ri is the growth rate constant of species i, p2 and
p3 refer to the probabilities of asymmetric (mis-) segregation of one
pair of Chr7 in the STICs and TMCs per cell division,
respectively. In general these probabilities depend on x, but for
simplicity we neglect such possible dependence. Notice that for the
TMCs, double mis-segregation of two pairs of Chr7 can result in
either 1+5 or 3+3, for which we assume an equal probability. For
cells with other Chr7 copy numbers, since their percentages are
very low, we neglected the even smaller contributions of possible
chromosome mis-segregation events. The governing rate equa-
tions are
dx1
dt~r1x1z2r2p2x2z
3
2r3p2
3x3
dx2
dt~r2(1{2p2)x2z3r3p3x3
dx3
dt~r3(1{3p3{
3
2p2
3)x3z2r2p2x2
dx4
dt~r4x4z3r3p3x3
dx5
dt~r5x5z
3
2r3p2
3x3
For convenience of discussion we also denote the percentage of
each subpopulation a at a given time point i as rai~xai=P
axai.
These quantities were what measured experimentally using FISH.
We consider three cases
N No mis-segregation, i.e., p2 = p3 = 0 SCs and MCs have no
direct mutual influence
N Mis-segregation exists, STICs and TMCs have no direct
mutual influences
N Missegragation exist, STICs partially inhibit the growth of
TMCs, which is modeled by a Hill function as
r3~r03z
D3
1z(r2=r2c)3, and MCs activate the growth of SCs,
which is modeled as r2~r02z
D2(r3=r3c)2
1z(r3=r3c)2, where r0
3zD3 and
r03z
1
2D3 are the growth rate constant of TMC when STIC
percentage r2~0 and r2~r2c, respectively, and r02 and
r02z
1
2D2 are the growth rate constant of STIC when TMC
percentage r3~0and r3~r3c, respectively. We actually also
consider the case with Hill coefficient 4 instead of 2, but the
result shows no significant change
Numerical method
The above ordinary differential equations were solved using
Matlab. At the beginning of each passage, we rescale xai so
Pa
xai~N0. Experimentally N0~5|105. For our mathematical
modeling the exact number of N0 does not affect the results. For
passage 1, we used the values of r used experimentally as the initial
values. For subsequent passages, we used the values r calculated at
the end of previous passage as the initial values.
For each case, the best set of parameters was obtained by
minimizing
R~Xa,i
(rcalai {rexp
ai )2
where rcalai and r
expai refer to the calculated and measured
percentages of subpopulation a at the end of passage i,
respectively. We used the down-hill simplex approach [28] to
perform the minimization. With each best set of parameters, we
predicted the doubling times.
Results
Tumor heterogeneity specified by Chr7-CNV commonlyexists in high and low grade gliomas
To determine Chr7 copy number variation (CNV) at the cell
subpopulation level, we performed fluorescent in situ hybridization
(FISH), with dual probes for the EGFR gene and the centromeric
region of chromosome 7 (CEP7). We examined GBM and
oligodendroglial tumor (OT), the second-most-common group of
gliomas, characterized by oligodendroglial features. OT includes
oligodendroglioma (OG, grade II), oligoastrocytoma (OA, grade
II); and anaplastic oligodendroglioma (AO, grade III), based on
criteria of the World Health Organization. The number of Chr7
centromeres per nucleus, detected by the FISH CEP7 probe, was
determined by counting over 250 cells per tumor, and these data
were used to establish the level of tumor heterogeneity with regard
to Chr7-CNV. We then performed a comparison of differences in
the equilibrium state for tumor heterogeneity based on Chr7-CNV
data from 14 GBMs and 12 OGs. There was a significantly higher
percentage of cells carrying more than 2 copies of Chr7
(amplification) in GBM compared to OG (P,0.0005)
(Figure 1A). In contrast to OG, the Chr7-CNV in OA and AO
was close to that of GBM, with representative data shown in
Figure 1B.
To determine if Chr7 CNV-characterized tumor cell popula-
tions are viable and contributing to the clonal diversity within each
tumor, we examined short-term (4–6 weeks with 1–2 passages)
primary cultures under serum adherent (SA) and/or neural sphere
(NS) culture conditions. We found subpopulation cells with Chr7-
CNV in all examined glioma primary cultures (Figure 1B). There
was a higher percentage of cells with Chr7-amplification in
cultures from GBM than in those from OG. In both cases, the
percentage of cells with Chr7-ampliciation was higher in primary
cultures than in the corresponding tumor. For AO, which is a
more progressive type of OT, we also observed a similar Chr7-
amplification in the tumor and in its derived primary culture.
Interestingly, in oligo-astro mixed OT, named ‘‘OA’’, the
equilibrium in cell composition for Chr7-heterogeneity in the
original tumors was similar to that in GBM. However, in OA-
derived primary cultures, we observed heterogeneity strikingly
resembling that of OG, with a majority of cells having two Chr7
copies and fewer than 40% of cells carrying three or more copies
of Chr7 (Figure 1B).
We then compared the patterns of Chr7-heterogeneity in OG
or GBM with recurrent and de novo status, and found no
correlation. However, there were incremental increases in the
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percentage of cells with Chr7-amplification in sequential GBMs
(i.e. tumors sampled over time at recurrence or progression) from a
patient with neurofibromatosis (Figure 1C). This is consistent with
the analysis above showing a faster growth capability for cells with
Chr7-amplificaiton.
A direct molecular consequence of Chr7 amplification is the
amplification of the oncogene EGFR residing within it, which
confers a growth advantage. Focal amplification of EGFR is
commonly seen in the classical subtype of GBM [29]. We thus
compared Chr7-CNV with EGFR-CNV, determined by real-time
comparative quantitative PCR. We found a balanced EGFR
relative to reference genes (ratio 0.7–1.3, given a 20–30%
variation in quantification) in all oligodendroglial tumors (n = 17)
and in 53% of the GBMs (n = 51), and a low level of EGFR
amplification (ratio 1.4–2) in 23.5% of GBMs, all well correlated
with calculated EGFR levels, based on the percentage of cells and
their Chr7 number. In the remaining 23.5% of GBM, we found a
high level of EGFR amplification (ratio between 5–48), which was
verified by EGFR/CEP7 FISH to be focal EGFR amplification
(Figure 1D). In EGFR (focal) amplified GBMs, the pattern of
Chr7-tumor heterogeneity was found to be similar to that in
GBMs without EGFR (focal) amplification.
Taken together, we observed a substantial level of tumor
heterogeneity, with cells showing Chr7-CNV commonly occurring
in both low- and high-grade gliomas. Monosomy of chromosome
10 is also a chromosome instability functionally related to tumor
malignancy [30] and occurs in about 80% of GBM tumors. When
present, monosomy 10 is shown homogenously, in contrast to the
heterogeneity seen for Chr7. This difference suggests that there
must be an active process for maintaining Chr7 heterogeneity in
tumors, which we have identified to be Chr7-MS. To further study
this process we examined Chr7-MS and Chr7-CNV in established
glioma cell lines.
Chr7-MS is involved in maintaining cell heterogeneity inestablished glioma cell lines
We performed B-banding using Giemsa stain on chromosome
spreads of six human malignant glioma cell lines and determined
the range of whole chromosome numbers (WCN) clustered around
the mode based on more than 7 cells. EGFR/CEP7 FISH were
performed and counts of CEP7 signals in more than 250
interphase cells were used to determine the percentage of cells
carrying different numbers of Chr7 (Figure 2A). All glioma cell
lines showed co-existence of diverse cell subpopulations based on
Figure 1. Proportion of cells with Chr7 number variation in high- and low-grade gliomas and glioma primary cultures. A, ternary plotof population proportions with 1 copy (deletion), 2 copies (normal), and 3 or more (amplification) copies of Chr7, based on CEP7 signals inFfuorescent in situ hybridization (FISH) of 14 glioblastoma multiformes (GBMs, Red triangle) and 12 oligodendroglial tumor (OGs, black square),P = 0.0012 from MANOVA analysis. B, comparison of tumor heterogeneity with regard to chromosome 7 (Chr7) aneuploidy in the original tumor (T)and corresponding 3–4 week-old primary cultures under serum adherent (SA) or neural sphere (NS) culture conditions. C–D, patterns of cells withChr7-CNV in sequential and high-EGFR amplified GBMs. Representative FISH pictures of cells carrying 1–4 copies of Chr7 with focal EGFRamplification.doi:10.1371/journal.pone.0080898.g001
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Chr7-CNV, with a significant percentage of cells carrying 2-, 3-,
and 4-copies of Chr7 with a near-diploid karyotype (U251, U87
and LG11), 4-, 5-, and 6-copies of Chr7 in near-triploids/
tetraploid (A172 and LN229), and 6-, 7-, and 8-copies of Chr7 in
near-pentaploid karyotypes (T98G).
Under SA-culture conditions that have been used to culture
these glioma cell lines, we established subcultures from single-cell
plating or single soft-agar colonies of the glioma cell lines, which
we named SA clones (SA1, SA2, etc). FISH showed re-appearance
of Chr7-cell heterogeneity in each of the SA clones (Figure 2B),
retaining characteristic features of Chr7 and EGFR amplification
and/or translocation that were found in their parental cultures
(Figure 2D). Interestingly, the percentage of those cells that had
been in the majority in the parental culture decreased in the clonal
SA subcultures. The exception was LN229, which was originally
composed of two subpopulations of cells nearly equal in
percentage. Evidently, tumor heterogeneity was maintained in
the established glioma cell line by Chr7-MS. It also indicates that
an established cell line is a cultured ecosystem with cell
subpopulations reaching certain equilibrium over time. We then
wondered if by changing culture conditions we could change the
heterogeneity equilibrium, such as to allow a previously minority
cell subpopulation to become dominant under new culture
conditions. If that proved to be the case, we would be able to
vary culture conditions to obtain sufficient numbers of the various
minority cells for further study of their phenotypes and contribu-
tions to overall growth.
We exposed glioma cell lines to NS culture conditions, which
were originally developed for culturing neural stem cells and then
modified for enriching glioma cells expressing neural stem-like cell
features [31,32]. We found that after a month’s culture in NS
conditions, during which there was a massive dying of cells, (with
the dead cells repeatedly removed by passing cells back and forth
between non-adherent and fibronectin-mediated adherent condi-
tions in NS medium), a minority cell subpopulation in the parental
line came to dominate the NS subcultures. We further established
clonal NS subcultures from single colonies formed in soft-agar
prepared using NS medium, and named these ‘‘NS clones’’ (NS1,
NS2, etc). Figure 2C shows representative FISH data of NS
subcultures of glioma cell lines. These data show re-appearances of
Chr7-cell heterogeneity from clonal NS subcultures.
It took 4 weeks for colonies to form in soft agar, prior to their
transfer to SA or NS culture conditions for further growth. After
transfer, it took about 2 weeks to obtain enough cells (26105) for
FISH analysis. The time required to re-establish culture hetero-
geneity from a single cell was less than 18 cell divisions, which took
about 6–7 weeks. Re-gaining of heterogeneous cell cultures with
Chr7-CNV in clonal SA and NS subcultures from all studied
Figure 2. Equilibrium of heterogeneity in cells with Chr7-CNV in established glioma cell lines and their clonal subcultures. A–B,percentage of cells with Chr7-CNV in glioma cell lines, and their SA and NS subcultures from single (e.g. 1, 2) or mixed (mix) soft-agar colonies,respectively. Whole chromosome numbers (WCN) ranging near the mode were found in .50% of cells in each glioma cell line. D, FISH picturesshowing normal (n) and derivative (d) Chr7 and the unknown (?) chromosome carrying a translocated EGFR from a representative metaphase cell foreach cell line. The chromosome is shown by DAPI (blue), centromere and EGFR are shown by FISH probes for CEP7 (green) and EGFR (red).doi:10.1371/journal.pone.0080898.g002
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glioma cell lines demonstrated that Chr7-MS could happen in any
subpopulation cell to drive tumor population diversity, while the
culture conditions determined the heterogeneity equilibrium of the
tumor cell types.
Dramatic changes in the equilibrium of cell subpopulations
between SA and NS cultures were noted for the two glioma cell
lines with near diploid karyotypes, U251 and U87. Whether
chromosome instability, here Chr7-MS, is an important mecha-
nism for generating/maintaining tumor/culture cell heterogeneity,
to the benefit of overall tumor growth, was the question we
attempted to address next by focusing on the characterization of
cell subpopulations of U251, as described below.
Distinct karyotypes in three cell subpopulations of U251There are different variants of U251 (see Table S2). The U251
used in this study was previous reported as U251HF, highly
tumorigenic and forming invasive intracranial (i.c.) xenografts that
displayed GBM histological hallmarks [33]. It has the DNA
microsatellite fingerprinting most similar to U251 in the NCI-60
cell line panel [34], carrying all short tandem repeat (STR)
markers but the one in the Y chromosome. Our FISH analysis
using CEPX/CEPY dual probes verified loss of the Y chromo-
some in U251HF. As for U251 (NCI), our U251 contains
homozygous mutations of PTEN [E242fs*15 (723 724 insTT)] and
TP53 (R273H), a derivative Chr7 with amplification of 7p and
deletion of 7q, and monosomy 10. In contrast to heterogeneity of
cells with Chr7-CNV (see Figure 2), 97% cells in U251 contains
one copy of chromosome 10 and two copies ‘‘mutant’’ PTEN
(Figure 3A, panel a).
By analyzing over a hundred metaphase cells of U251 and its
derived SA and NS clones, we found that 91% of the cells were
near diploid, carrying 1-, 2- and 3-copies of Chr7 and a small
percentage cells were near tetraploid, carrying 4-, 5- and 6-copies
of Chr7. In the parental culture, the majority cell type had two
normal and one abnormal 7q-deleted Chr7 (designated as
3Chr7:2n,1d) and two minority subpopulations of cells carrying
either two normal Chr7s (2Chr7:2n) or one normal and one 7q-
deleted Chr7 (2Chr7:1n,1d) (Figure 3A). The percentage of
2Chr7:1n,1d cells increased in clonal SA subcultures (Figure 2B),
and further increased after 1 month in culture under NS
conditions (see SA1-NS in Figure 3B). Percentage of 2Chr7:1n,1d
cells was as high as 90–92% in NS clones (see NS1 in Figure 3B),
as far as they were maintained in NS conditions. The 2Chr7:2n
cells, however, remained a minor subpopulation in parental, SA
and NS subcultures.
In an initially unrelated experiment involving use of lentiviral-
mediated expression of EFEMP1, we used U251 parental cells to
examine doxycyclin-induced expression of ectopic EFEMP1 (‘‘P-
E1’’ cultures). We found that the 2Chr7:2n cell percentage
increased to nearly 80% in P-E1; whereas, cells infected with
control vector (see P-Vec in Figure 3B) had a Chr7-cell population
equilibrium similar to the U251 parental line, with 3Chr7:2n,1d
cells as majority subpopulation (Figure 3B). We have previously
reported on the tumor suppressive effect of EFEMP1 in GBM and
EFEMP1’s function in reducing the EGFR signaling activities in
glioma cells [35]. The observed reduction in the proportion of
3Chr7:2n,1d cells in U251from ectopic expression of EFEMP1 is a
new finding, consistent with EFEMP19s suppression of EGFR
signaling activity, which is supported by subsequently obtained
data showing a high level of EGFR expression in 3Chr7:2n,1d
cells (Figure 4B). We then examined the cell composition in P-E1
cultures after withdrawing the doxycyclin-induced EFEMP1
expression (P-E1wd culture). Figure 3B shows that after two
passages without EFEMP1 induction (indicated by completely
gone of RFP expression), the majority of cells in the P-E1wd
culture remained the 2Chr7:2n cells, with a percentage as high as
80%. This observation indicates the robustness of these culture
compositions once equilibrium in cell subpopulations has been
established.
Chr7-MS is responsible for inter-conversion ofsubpopulation cells
We performed comparative genome hybridization (CGH) to
determine if there are other DNA-level alterations besides Chr7
that could be specific to certain Chr7-subpopulation cells in U251.
If we found such characteristic CNV, it could be used to trace the
origins of Chr7 aneuploid cells as coming either from the parental
culture or arising de novo by Chr7-MS. We compared CGH profiles
of U251 parental, NS1, and SA1 re-selected to enrich NS cells by
culturing in NS-condiiton for 4 weeks (named as SA1-NS). It is
clear that the majority cells in the parental and parentally derived
NS subcultures carry one 7q-deleted Chr7 with a nearly complete
q-arm deletion and amplification of the distal p-arm (Figure 3C).
Figure 3C further demonstrates that the majority cells of the
parental culture also carried two copies of normal Chr7, while
both NS subcultures derived from parental and SA1 carried one
normal Chr7 (the other being a 7q-deleted Chr7), which is
consistent with the results of FISH (Figure 2D and Figure 3A).
Comparison of CNV in other chromosomes showed regional
amplifications in chromosomes 8, 17, and 22 that were found
specifically in NS1 (majority = 2Chr7:1n,1d cells, Figure 3D,
middle panel), but were absent in U251 parental (majori-
ty = 2Chr7:2n,1d cells) and SA1-NS (majority = 2Chr7:1n,1d cells)
(Figure 3D, left and right panels, respectively). In contrast, the
CGH profiles of U251 parental and SA1-NS were highly similar
for all chromosomes but Chr7. Clearly, the 2Chr7:1n,1d cells in
NS1 are descendents of 2Chr7:1n,1d cells pre-existing as the
original minority cell subpopulation in U251, while the
2Chr7:1n,1d cells in SA1-NS are descendents of a 3-Chr7:2n,1d
cell in U251. The common difference between parental and SA1-
NS is the loss of one normal copy of Chr7. These results clearly
show that Chr7-MS was responsible for converting a 3Chr7:2n,1d
cell into a 2Chr7:1n,1d cell, thereby restoring cell heterogeneity in
the glioma culture. The reverse case, where Chr7-MS converts a
2Chr7:1n,1d cell into 3Chr7:2n,1d cell has also been demonstrat-
ed by FISH and the functional assays described below.
Cell subpopulations in U251 have distinct phenotypesThrough our research on U251 and its NS subcultures and
EFEMP1-infectants, we obtained three syngeneic cultures, each
having a different cell subpopulation type in majority, these being
3Chr7:2n,1d, 2Chr7:1n,1d and 2Chr7:2n cells in parental (P),
mixed or clonal NS subcultures (NS, NS1, NS2), and EFEMP1-
infectants withdraw the induction of transgene expression (P-
E1wd), respectively (Figure 3B). These syngeneic cultures provided
us with a unique resource for conducting a study on the
phenotypic diversity of subpopulation cells and the benefit a
tumor derives from having such cell composition diversity.
Important here are the mechanisms that control the dynamics of
tumor cell population equilibrium, which could aid our under-
standing of cancer plasticity and failures in GBM treatment.
First we carried out a comparison of the expression of genes/
proteins reported to mark neural stem cells, and to cause changes
in cancer cell invasive, proliferative and angiogenic behaviors. We
limited molecular analysis to parental (majority = 3Chr7:2n,1d
cells) and NS subcultures (majority = 2Chr7:1n,1d cells). Com-
pared to 3Chr7:2n,1d cells, the 2Chr7:1n,1d cells expressed genes
of neural stem cells (PROM1, BMI1, MELK, MSI1). Immunocyto-
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fluorescence of NS1 cells with primary antibodies for CD133,
MELK, and BMI1 showed positive staining in the majority cells
for MELK and BMI1, but very few cells stained by CD133 (Figure
S1), which is consistent with a low expression of CD133 in NS
subcultures compared to no expression in parental cells. The
2Chr7:1n,1d cells exhibited a more invasive phenotype by
expressing higher levels of the pro-invasive genes (IGFBP2,
PDGFRA, RHOC, FOXM1, and PLAU) and matrix metalloprotei-
nase 2 (MMP2) (Figure 4A), all of which are well-reported for
cancers, including GBM [36–38]. In contrast, the 3Chr7:2n,1d
cells demonstrated a proliferative phenotype with secretion of pro-
angiogenic proteins (VEGFA and SPP1) and expression of EGFR
(Figure 4A–B) at significantly high levels compared to that by
2Chr7:1n,1d cells.
We then examined the in vitro growth of the three syngeneic
cultures. Because it is the change of culture medium and
attachment that allowed the establishment of NS subcultures with
enrichment of cells originally in a low percentage in the parental
Figure 3. Distinct karyotypes of three subpopulation cells in U251. A, representative metaphase FISH pictures of PTEN/CEP10 and EGFR/CEP7 dual probes showing all cells carrying one copy of Chr10, an unknown chromosome with a PTEN translocation, and three cell types differing intheir composition of normal and derivative Chr7 (dChr7). Arrow points to dChr7; arrowhead to normal Chr7. B, percentage of majority cells in theparental culture, derived or converted SA or NS subcultures, and the parental culture after lentiviral transductions by pTRIPZ-Vec (P-Vec), pTRIPZ-EFEMP1 with (P-E1) or after withdrawal (P-E1wd) of doxycyclin. C–D, comparison of DNA copy number variation in chromosomes 7, 8, 17, and 22 forU251 parental derived or converted NS subcultures of NS1 or SA1-NS, respectively. The Y axis is the log ratio of intensity (the ratio of test sample andnormal blood) from comparative genome hybridization. Amplifications or deletions are shown by blue lines above or below the red or green areas,respectively, based on Z-score, and those with marked changes are highlighted in purple.doi:10.1371/journal.pone.0080898.g003
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Figure 4. Distinct phenotypes of subpopulation cells in U251. A, real-time qRT-PCR (right) quantification of the expression of genesassociated with neural stem cell features as well as glioma cell migration and invasion, normalized to ACTB, in cells of the same cultures used forzymography, and enzyme immunometric assays (left) for quantification of VEGFA (VEGF-165) and SPP1 (Osteopontin) in conditioned medium,normalized by cell numbers. Bar and line height are mean and SD based on quantification of 3–6 sets of independent cultures. B, western blot (top)and immunocytofluorescence (bottom) of EGFR (1:1000 from Cell Signaling) in U251 parental (P) and two clonal NS lines (NS1 and NS2). C, soft agarcolony formation assay of U251 parental (P), NS1, and P-E1wd lines. D, s.c. tumorigenicity assay of cells described above, with follow-up of tumorgrowth as described previously [33]. E, immunocytofluorescence analysis of NS1 and SA1-NS before and after being subjected to neural stem celldifferentiation conditions described in Methods.doi:10.1371/journal.pone.0080898.g004
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culture, we used a soft agar colony formation assay to determine
each subpopulation’s anchorage-independent growth in serum-
and/or EGF/bFGF-containing media. All three syngeneic cul-
tures formed colonies at a similar rate in soft agar with medium
containing serum (Figure 4C). However, only NS1 (majori-
ty = 2Chr7:1n,1d) formed colonies in serum-free NS culture
medium, while the P (majority = 3Chr7:2n,1d) and P-E1wd
(majority = 2Chr7:2n) failed. This indicated a similar serum-
dependent, anchorage independent growth phenotype for
3Chr7:2n,1d and 2Chr7:2n cells, while 2Chr7:1n,1d cells were
more flexible with regard to growth conditions.
We carried out subcutaneous (s.c.) implantation of glioma cells
(16106) with 10 independent implantations to examine tumor
onset and growth, which are dependent on tumor-cell-induced
angiogenesis [35]. Both P (majority = 3Chr7:2n,1d) and P-E1wd
(majority = 2Chr7:2n) are tumorigenic in the s.c. xenograft model,
with the former showing faster tumor growth than the latter
(Figure 4D). In contrast, NS1 (majority = 2Chr7:1n,1d) failed to
form s.c. xenografts, even with the follow-up time extended to 44
days. A repeat of the implantation with cells prepared indepen-
dently at a different time also failed to give a xenograft tumor.
Overall, the results from assays on in vitro molecular profiles and
growth, and in vivo s.c. tumorigenicity, showed distinct growth,
angiogenesis, and invasion features that were consistent for each of
the subpopulation cell types in U251, with 3Chr7:2n,1d and
2Chr7:2n cells sharing similarity in their serum-dependent growth
and ability to form s.c. xenografts, while the 2Chr7:1n,1d cells
expressed more invasive proteins and was unable to form s.c.
xenografts. The above described neural stem marker expression,
sphere-forming phenotype, and the following described re-
establishment of tumor hierarchy by a U251-NS clonal line are
consistent with features expected from tumor cells with stem-like
properties, identified from primary glioma by NS culture [31] or
CD133-antibody mediated cell sorting techniques [39]. The high
i.c. tumorigenecity for 2Chr7:1n,1d cells further defines its feature
as tumor initiating cell (TIC). Other stem-like properties of
2Chr7:1n,1d cells include the stem-like cell multipotency shown in
neural stem cell differentiation assay in vitro and endothelial
differentiation in vivo. The former feature was demonstrated by
both NS subcultures of parental-line origin or newly generated by
Chr7-MS. Both showed increase in expression of glial cell marker
GFAP and neuronal cell marker MAP2, and decrease in
expression of neural stem cell marker NES, following 1-2 weeks
of culturing in differentiation conditions. This expression pheno-
type was in marked contrast to that of the undifferentiated cells
analyzed prior to subjecting them to the differentiation conditions
(Figure 4E). The ability of U251-NS cell in forming blood vessel
was also shown in below described i.c. xenograft (Figure 5). Hence
2Chr7:1n,1d cell are the stem-like TIC (STIC), the cell
subpopulations within primary cultures of malignant glioma with
a long-term self-renewal capacity [40].
We also evaluated colony formation rate for both subpopulation
cells in U251 by single cell plating of parental cells in adherent
culture, limiting dilution assays for NS subcultures in NS and soft
agar conditions. The colony formation rate for U251 was 52+/
28% from plating an average of 0.5, 0.7 and 1 cell per well. The
sphere formation rate for U251-NS1 was 74%+/23% from
limiting dilution (100, 20, 5 and 1 cell). The colony formation rate
in soft agar for U251-NS1 was 6–7% (see Figure 4C). Because NS1
originated from a single soft agar colony, the ability to re-form
colonies verified an inherited self-renewing ability, which is
frequently referred to as stemness for cancer stem cells. Here
our characterization of U251 showed diverse phenotypes of
subpopulation cells, which are able to self-renew and inter-convert
via Chr7-MS.
Tumor growth benefits from having cell subpopulationswith diverse phenotypes
We further examined the phenotypic characteristics of the
STIC from U251 to determine their ability to form tumors in the
intracranial (i.c.) xenograft model, to restore subpopulation cell
heterogeneity, and to form blood vessels by endothelial trans-
differentiation, as previously described for STIC [41,42]. NS1,
which lacked s.c. tumorigenicity from implantation with 16106
cells (see Figure 4D), formed i.c. xenografts from implantation with
16104 or 16105 cells (Figure 5A). FISH analysis of the resulting
i.c. xenografts showed a marked increase in the percentage of cells
carrying 1 and 3 copies of Chr7 (Figure 5B). The cells with 1 and
3-copies of Chr7 could be found physically near each other,
suggesting Chr7-MS of STIC during i.c. xenograft formation.
To demonstrate the in vivo infiltrative feature of STIC from
U251 shown by in vitro assays, we infected U251 parental and NS
cells under semi-confluent conditions with two lentiviral vectors,
pGIPZ and pTRIPZ, that express green (GFP) and red (RFP)
fluorescent proteins, respectively. After 1–2 weeks of culturing,
with puromycin elimination of non-infected cells, the infected cells
were pooled together for i.c. co-implantation, alone or mixed at
various ratios. As shown in Figure 5C, the i.c. xenografts derived
from co-implantation with 10% GFP and 90% RFP cells showed a
majority of cells expressing GFP and these were located in the
center of the tumor mass, whereas cells expressing RFP were
found at the peritumoral boundaries. Reversing the co-implanta-
tion percentages resulted in an even more striking separation of
these two subpopulations, with most of the RFP cells found in the
infiltrating tumor boundary. Immunofluorescence analysis verified
that the infiltrating RFP cells expressed BMI1 and MELK
(Figure 5D), the same as shown in their in vitro NS cultures
(Figure 4A). These genes have been reported to express in STICs
[32].
In i.c. xenografts from co-implantation with 1% RFP and 99%
GFP cells, some RFP-expressing cells were associated with blood
vessels forming within the main tumor mass_ENREF_10. The
presence of naturally autofluorescent erythrocytes[43] in these
vascular channels confirmed that they were functionally active,
vascular channels formed by tumor cells. Such vessels are
commonly found, and are considered to be a pathologic
vascularization mechanism in GBM [44,45]. The RFP cells
expressed CD133, a marker frequently used to identify brain-
tumor stem-like cells [39], as well as the glioma-associated,
secreted protein SPARC [46] (Figure 5E), and the endothelial cell
marker CD31, as revealed by confocal microscopy (Figure 5F left).
These observations demonstrated endothelial trans-differentiation
of STIC from U251, which maintained their glial cell character-
istics as shown by expression of GFAP (Figure 5F right).
The above described i.c. xenograft experiments showed the
tumor mass-forming cell (TMC) feature in majority cells of U251,
which was in striking contrast to less proliferative more invasive
phenotype shown in STIC from U251. Such functional diversity
between two glioma cell subpopulations with Chr7-CNV would be
synergistic to overall tumor growth. Indeed, by analyzing animal
survival time in relation to the proportions of U251 subpopulaiton
cells with STIC and TMC features, it was found that a ratio of
approximately 1 STIC: 2 TMC had the most deleterious effect on
mouse survival (Figure 5G). Interestingly, this is also the ratio that
showed the highest overall growth speed in vitro under SA
conditions (Figure 6B), from studying the dynamics of cell
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composition from homogeneity to heterogeneity, as detailed in the
following section.
The overall results described above demonstrated phenotypic
differences between TMC and STIC subpopulations of U251.
CGH data showed one copy Chr7 and regional amplification in
chromosomes 8, 17, and 22 as the major DNA level differences
between parental (majority TMC) and NS1 (majority STIC). It is
not determined by this study if alterations in chromosomes 8, 17,
and 22 are responsible for the different tumor formation
properties. However, combining the data of the CGH and the
neural stem cell differentiation assays for NS1 and SA1-NS, their
involvement in STIC features was excluded.
Mathematical modeling of cell equilibrium fromhomogeneity to heterogeneity
By changing culture conditions to NS conditions that favor
glioma cells carrying STIC features, we were able to maintain a
nearly homogenous culture with STIC, which was a very minor
subpopulation in the U251 maintained under SA conditions.
Following the observation of autonomous Chr7-MS in single-cell-
derived subcultures, which restored the cell heterogeneity seen for
the parental culture (Figure 2), we examined the process of
restoring culture heterogeneity by returning NS1 with nearly
homogenous STIC to SA conditions, taking measurements of cell
doubling time and using FISH to analyze the proportion of cells
carrying different numbers of Chr7, over 29, 3-day, serial
passages. This gives a total of approximately 90 cell divisions.
This experiment was repeated once, with averages in cell doubling
time and percentage cells varying in Chr7-CNV applied in
mathematical modeling, to identify various parameters yielding
the observed changes in overall cell growth rate and the
subpopulation cell equilibrium. Cell proportions were determined
from the FISH data collected for more than 250 cells.
For simplicity, we did not distinguish among cells with different
normal and abnormal Chr7 compositions (Figure 6A). A set of
mathematical equations describe the time evolution of subpopu-
lations with 1–5 copies of Chr7. Because 92% of cells in NS1 were
2Chr7:1n,1d cells functionally defined as STICs, we considered
cells measured by FISH to carrying two copies of Chr7 to be
STICs. We called cells carrying three copies of Chr7 in U251 as
TMCs, based on their phenotypic characteristics described above
and the fact that metaphase FISH data did not show other
assortments of normal and abnormal Chr7 (we assumed such cells
were possible from Chr7-MS but were unable to survive or grow).
For analysis of cell-cell interactions in culture under SA conditions,
we ignored the small number of cells in U251 carrying 1 copy of
Chr7, with 1Chr7:1d cells observed in metaphase FISH.
We first considered the simplest model (model 1) with no inter-
conversion between subpopulations. This model predicted that the
subpopulation with the highest growth rate (i.e., TMCs) eventually
Figure 5. Distinct ‘‘go’’ and ‘‘grow’’ features of two cell subpopulations in U251 with optimal equilibrium benefiting overall tumorgrowth. A, H&E images (2X) and FISH images (100X) of i.c. xenografts derived from NS1. Arrowhead, double and single arrow point to cells with 1, 2,and 3 copies of Chr7, respectively. B, comparison of cell population equilibrium in NS1 (in vitro culture) and the derived i.c. tumors. C, fluorescenceimages of i.c. xenografts derived from co-implantation of RFP-labeled STIC-enriched U251-NS and GFP-labeled U251 in a 9:1 ratio. D–E,immunofluorescence images of i.c. xenografts from co-implantation of the two lines in a 1:99 ratio, with purple color marking BMI1 and MELKexpression by RFP cells at the tumor boundary and their expression of CD133 and SPARC in vascular mimicry within the bulk tumor mass. F, confocalimmunofluorescence images of i.c. xenografts derived from 100% RFP cells, with yellow color marking co-localization of RFP with CD31 or GFAP. G,Kaplan-Meier survival curves of mice after implanting a mixture of U251 parental and STIC-enriched NS subculture. Adjusted Hazard ratios (HRs) fromthe stratified analysis and p-value are from Cox regression analyses examining the effect of STIC percentage on survival.doi:10.1371/journal.pone.0080898.g005
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would sweep away other subpopulations (Figure 6B), which is
inconsistent with the experimental observations. We then
expanded the model to allow inter-conversions among subpopu-
lations through Chr7-MS (model 2). This model satisfactorily
reproduced the observed heterogeneity. Even though the TMC
had the fastest growth rate, with Chr7-MS, they always generated
other subpopulations, and the culture eventually approached a
steady-state distribution.
We experimentally tested the model for its power in predicting
changes in cell doubling time over serial passages. There were
qualitative differences between the experimental results and
outcomes predicted by either model 1 or model 2 (Figure 6C).
Therefore we further considered the possibility of interactions
between subpopulations. By considering Chr7-MS and interac-
tions between STIC and TMC, specifically, TMC enhancing
STIC growth, or STIC inhibiting TMC growth, or both, (model
3), we successfully reproduced the evolution of the subpopulation
proportions as for model 2, but further predicted the biphasic
behavior of the doubling time (Figure 6C). The nature of the
interactions between the subpopulations is not known and would
need further study to identify.
Figure 6. Mathematical modeling of experimental data with changes in population equilibrium from homogeneity toheterogeneity. A, schematic illustration of the working mechanism with Chr7-MS resulting in heterogeneous subpopulations and cellularphenotype inter-conversion, with STIC inhibiting growth of TMC and/or TMC stimulating growth of STIC. The Chr7 composition was shown forrepresentative subpopulation cells in U251, based on metaphase FISH.A higher growth rate for TMC (r3) was shown compared to that for STIC (r2).Cells marked by black circles were seen in metaphase FISH analysis, suggesting their ability to grow in vitro. Cells marked by gray boxes were not seenin metaphase FISH analysis, suggesting they are unable, or have very low ability to grow in vitro. B, changes in population equilibrium fromhomogeneity to heterogeneity in NS1 after serial, three-day passages in SA-culture conditions, using the same cell plating density (56105/100 mmdish), with cell types determined by FISH (experimental) and then modeled using different parameters as detailed in Methods. C. changes in cellgrowth speed as measured or predicted by various mathematical models. D, s.c. tumorigenicity assay showing the TMC features of 3Chr7:2n,1d cellsconverted from STIC by increasing percentage tumor onset (a tumor size of ,50 mm3). The plot with percent uses percents computed via theKaplan-Meier method. Log-rank test for trend with 3Chr7:2n,1d cells shows two-sided P,0.0001.doi:10.1371/journal.pone.0080898.g006
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The parameters that best fit the dynamic cell population
equilibrium measured by CEP7 FISH were shown in Table 1. It
showed a higher growth rate of TMC than STIC, suggesting that
SA culture conditions favor TMC growth, but Ch7-MS prevented
its homogenizing the culture over time. Our model only assumed
Chr7-MS, since the experimental data provided only cells with
Chr7-CNV. The possibility of other chromosome mis-segregaiton
is certainly there, just not being measured. The mis-segregation
rates of Chr7-MS in our U251 model are very low (,0.001 and
,0.01 per cell division, respectively), which are in the range of
aneuploidy rates reported in human cancer cells [47] and yeast
[48].
Given that Chr7-MS is a mechanism for generating culture
heterogeneity, the model predicted that a cell colony formed from
a single TMC carrying two normal and one deleted Chr7 should
also generate a small subpopulation of cells carrying just two
normal Chr7 copies (i.e. 2Chr7:2n), in addition to give rise STIC
(2Chr7:1n,1d) as shown in Figure 6A. This was indeed proven to
be the case by metaphase FISH analysis of single-cell derived
clones of U251. These cells were shown to be less responsive to
EFEMP1-mediated suppression compared to TMC
(3Chr7:1n,1d), which allowed their dominating the culture after
a sustained period of culture with expression of ectopic EFEMP1
(see P-E1 in Figure 3B). Overall, the data showed the complexity
of cancer cell phenotypes that could be enhanced by the existence
of chromosomes with structural abnormality, in addition to their
mis-segregations.
Phenotypic similarity between TMC in the parentalculture and that derived from STIC
We have shown above inter-conversion of subpopulation cells in
U251 by Chr7-MS, with the STIC phenotype restored by loss of
one normal copy of Chr7 from TMC. We also saw the converse,
where TMC appeared in culture derived from a single STIC in
NS1, whereas the percentage TMC increased after culturing in SA
conditions (Figure 6B). To determine if the gain of one copy of
Chr7 in STIC was enough to restore the TMC phenotype, we
examined the s.c. tumorigenicity of NS1 after it was subjected to
SA culture conditions, where the percentage of new STIC
increased, based on the discovery of STIC lacking s.c. tumor-
igenecity (Figure 4D).
We s.c. implanted NS1 after first culturing them under SA
conditions for differing numbers of passages (0, 5, 9, 24). FISH
showed that the percentages of TMC increased with increase of
passage numbers, from 1%, to 3%, 24%, and 61%, respectively.
The s.c. xenograft volume measured after implantation (16106
cells, total 10 implantations) correlated positively with the
percentage of TMC prior to implantation (P,0.0001)
(Figure 6D). Three weeks after implantation, the cell population
subjected to over 9 passages under SA conditions, which contained
more that 24% TMC, behaved the same as the parental culture in
regards to tumor onset (once a tumor reached approximately
50 mm3 in size, usually a fast tumor growth will start). In contrast,
cells passed only 5 times under SA conditions, which contained
fewer than 5% 3Chr7:2n,1d cells, had poor tumor onset. Clearly,
the gain of one normal copy of Chr7 by some cells in the STIC
population through Chr7-MS restored the TMC phenotype.
VEGFA overexpression enabled s.c. tumorigenecity ofSTIC
Failure of NS1 to form tumors in the s.c. xenograft model at a
high cell dose (16106 cells) is in striking contrast to the high i.c.
tumorigenecity from lower number of cell (tested on 16103,
16104, 16105 cells). This demonstrates the requirement of
orthotopic environment for STIC to grow in the first place. The
result of rescuing STIC in s.c. tumorigenecity by a large number of
TMC suggests quantitative improvement of tumor microenviron-
ment by TMC. From the observations that overexpression of
VEGFA restores s.c. tumorigenecity of U251 that was suppressed
by EFEMP1 [35] and that TMC secretes a high level of VEGFA
(Figure 4A), we hypothesized that VEGFA overexpression may
enable s.c. tumorigenecity of STIC. This hypothesis was supported
by results shown in Figure 7, with a high s.c. tumoirgenecity in
VEGF-165 transfected NS1.
Irradiation enhances chromosome mis-segregation ofglioma cells
Radiation is a frontline therapy for gliomas, although it is
known to cause various stress responses in the treated cells. We
used the defined U251 model and FISH technology to determine
if radiation increased the Chr7-MS rate. To provide proof-of-
concept data, we performed a simple experiment by exposing
semi-confluent U251 cells to single, acute-dose applications at 2
and 5 Gy, and fixing treated cells 24 hours later, for subsequent
FISH analysis. The radiation treatments caused an increase in the
percentage of cells carrying one or two copies of Chr7, at the
expense of cells with three copies of Chr7 (Figure 8). Because the
U251 cell doubling time is about 22 hours, the acute radiation
effect on changes in the proportions of cell subpopulations suggests
an increase in the Chr7-MS rate in the dividing, majority, TMCs.
Consistently, metaphase chromosome spreads showed increases of
both 2Chr7:2n and 2Chr7:1n,1d cells after irradiation. Taken
together with information about the radioresistance of glioma
stem-like cells [49] and STIC features of 2Chr7:1n,1d cells
described above, our findings describe a new mechanism for
explaining glioma radio-resistance and tumor recurrence that is
worthy of further investigation.
Discussion
Non-random distributions of chromosomal gains and losses are
common in clinical tumors at both early and late stages, and these
are maintained in metastases and cell lines derived from primary
tumors. These observations are consistent with an interpretation of
Table 1. Parameters that best fit of the percentage time course data.
r1 r20 D2 r2c r30 D3 r3c r4 r5 p2 p3 Rmin
Model 1 1.6 1.6 0 N/A 1.8 0 N/A 1.6 0.02 0 0 0.5
Model 2 1.5 1.6 0 N/A 1.9 0 N/A 0.2 0.1 0.002 0.02 0.1
Model 3 1.8 1.7 0.3 0.2 2.1 0.7 0.4 0.05 0.01 0.001 0.01 0.1
The parameter r20 is chosen to have an initial doubling time of 1.1 days.doi:10.1371/journal.pone.0080898.t001
Chromosome Mis-Segregation and Tumor Heterogeneity
PLOS ONE | www.plosone.org 13 November 2013 | Volume 8 | Issue 11 | e80898
chromosome instability as a driver of cancer evolution, progres-
sion, and drug resistance through the creation of variable
karyotypes, most of them likely inviable, but with selection for
the quasi-stable cancer karyotypes that remain [50,51]. The
catalytic role of chromosome instability in cancer development has
also been suggested by a theoretical study of cancer progression
[52]. It remains an open question in cancer biology whether
chromosome instability is involved in maintenance of tumor
heterogeneity, defined by cell subpopulations having specific
chromosome gains and losses. Our study showed a common
existence of Chr7-aneuploid cell subpopulations within gliomas of
various malignancies, glioma primary cultures, and cell lines.
Finding the re-appearance of specific Chr7-defined cell subpop-
ulations in all single-cell-derived subcultures, of all examined
glioma cell lines, provided strong evidence for the involvement of
Chr7-MS in the maintenance of tumor heterogeneity in gliomas.
Importantly, by molecular and functional characterization of
U251 syngeneic cultures dominated by different cell subpopula-
tions, we discovered a phenotypic divergence of glioma cell
subpopulations following re-assortment of Chr7. Interestingly, the
behaviors of one subpopulation fit all criteria described for STIC,
especially for their pleiotropic cell feature in forming blood vessels
[31,39,41,42], in addition to their infiltrative behavior and ability
to convert into a highly proliferative cell phenotype by Chr7-MS.
The consequence of cell conversion from TMC to STIC by gain
an additional Chr7 from Chr7-MS, obviously causes drastic
changes in transcription. Comparing to TMC, we have shown
STIC to have higher invasiveness, which was correlated with a
higher level of expression of pro-invasive genes (Figure 4A), and a
peritumoral localization (Figure 5C). The phenotypic transition of
cells into a more invasive phenotype has been described as
epithelial-mesenchymal transition (EMT), which can be caused by
increasing the expression of EMT-responsive genes in both normal
and cancer cells. Here, in U251, we show that Chr7-MS causes a
similar phenotypic change.
However, the finding of STIC-promoted angiogenesis by Bao et
al [53] from studying matched CD133+ and CD1332 tumor cell
populations cultured from D456MG xenografts is not supported
by our results from studying functionally defined STIC and TMC
from U251. Our finding of VEGFA in promoting tumorigenesis of
STIC via angiogenesis is consistent with findings of Oka et al. [54]
by studying a line of multipotent, self-renewing cells derived from
fresh human GBM. We further showed with evidence from both
experimental study and statistical analysis that the angiogenesis
could come from proangiogenic factors made by TMCs.
Differential expression of EGFR has been shown in GBM
subpopulation cells that were tumorigenic, and increases in EGFR
level was shown to be responsible for the highly tumorigenic
property [55]. In TMC of U251, overexpression of anti-EGFR
protein EFEMP1 almost eliminated the TMC subpopulation
during in vitro culture of U251-E1 (see Figure 3B). However,
activation of the EGFR-mediated growth signal appears not be
used by STIC in U251. Activation of developmental signaling
pathways of Notch and hedgehog has been shown for STIC
[56,57]. Our finding of a higher NOTCH1 expression in STIC
compared to TMC of U251 (data not shown) needs to be further
explored to determine if STIC from U251 uses Notch signaling to
maintain its growth.
Overall functional characterizations of STIC and TMC
subpopulations in U251 provide convincing evidence that the
tumor benefits from carrying heterogeneous cell populations. It
also suggests that in glioblastoma, both the stem-like cells and
mass-forming cells may have the capacity to regenerate the other
population through Ch7-MS. This is a much more insidious and
complex capability than that originally assumed of a one-way
conversion of tumor stem-like cells to tumor mass cells. In having
Figure 7. Re-examination of U251-NS1 cell s.c. tumorigenicity after overexpression of VEGFA. U251-NS1 infected with retrovirual vectorsof VEGF-165 and LacZ described previously [35] were s.c. implanted in nude mice as described in Materials and Methods.doi:10.1371/journal.pone.0080898.g007
Figure 8. Effect of radiation on Chr7-MS. FISH was performed 1day after exposure to c-rays, in single dose, at the 2 and 5 Gy levels.doi:10.1371/journal.pone.0080898.g008
Chromosome Mis-Segregation and Tumor Heterogeneity
PLOS ONE | www.plosone.org 14 November 2013 | Volume 8 | Issue 11 | e80898
an invasive, pleiotropic cancer cell type, and a less invasive cancer
cell type that functions primarily to form the tumor mass, together
with cell type inter-conversion by mis-segregation of a specific
chromosome, the data we present here support the Darwinian
cancer evolution theory.
The finding of interchangeability among cancer cell subpopu-
lations by chromosome instability greatly helps to explain cancer
plasticity and robustness. Based on the Darwinian cancer
evolution theory, cancer benefits from the co-existence of diverse
cell subpopulations within the tumor. The presence of clonal
diversity has been shown to predict cancer progression in
esophageal adenocarcinoma [3]. Further, the synergistic effect of
cell subpopulation diversity on overall tumor growth is evident
from the co-implantation experiments using glioma cells express-
ing the EGFR deletion mutant and wild-type EGFR [58]. Both our
in vitro and in vivo experimental data demonstrated the existence of
an optimal equilibrium of tumor heterogeneity to overall growth,
which was surprisingly similar in the serum adherent in vitro culture
and the brain tumor orthotopic environment, between the invasive
stem-like cells and the proliferative nonstem-like cells. In addition
to the experimental data we presented, our mathematical
modeling of the experimental data supports the involvement of
chromosome instability in opposing clonal homogenization.
Furthermore, it suggests that interactions between the cell
subpopulations generated a benefit to overall cell growth, which
was not directly evident in the experimental data.
Overall, what we have presented here is that Chr7-MS in
glioma maintains tumor heterogeneity, which favors overall tumor
growth. The possibility of mis-segregation for chromosomes other
than Chr7 certainly exists; however, this was not examined during
our study. It would be interesting to determine whether there are
any additional chromosome mis-segregations involving different
chromosomes that also correlate with our observed cell phenotype
differences. We believe that our present work opens several new
questions to pursue, the answers to which would greatly advance
our understanding of cancer evolution, progression and resistance
to therapy.
Supporting Information
Figure S1 Characterization of stem cell marker expression in
U251-NS1 cells by immunocytofluorescence in undifferentiated
conditions described in Materials and Methods.
(TIF)
Table S1 9-STR DNA Profile of human glioma cell lines used in
this study.
(DOC)
Table S2 Comparison of U251 variants by 9 short tandem
repeat (STR) markers.
(DOC)
Acknowledgments
We acknowledge Dr. John van der Meer for editing.
Author Contributions
Conceived and designed the experiments: YZ. Performed the experiments:
YH NR HX AC NH CK FY JN ZL LY XZ YZ RG. Analyzed the data:
YH XT HZ ES ZJ MJ CL ML JX YZ. Contributed reagents/materials/
analysis tools: HX LY MJ CL ML JX YZ. Wrote the paper: ML JX YZ.
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